药学学报

Characteristics	Monolayer cell model	Stereo cell model	Organoid model
Advantages	1. Simple training 2. Model establishment time is short 3. Get results quickly 4. The model is easy to replicate	1. Maintain the material structure basis of the cellular microenvironment in the body 2. Some resistance to the growing environment 3. Cell-to-extracellular matrix interaction 4. The function of the cell can be expressed	1. Achieve similar natural organizational structure and function 2. Effectively reconstruct human tissue and have close physiology and pathophysiological status in vitro 3. Have some resistance to the growing environment 4. Has cell and extracellular matrix interactions 5. The function of the cell can be fully expressed
Disadvantages	1. Sensitive 2. High requirements for growth environment 3. Easy to be affected by many factors 4. Lack of cell and extracellular matrix interactions 5. Cell function cannot be fully expressed	1. Cultivation is more complicated 2. Model establishment time is longer 3. The result is slow 4. The model success rate is not high	1. Cultivate difficulties 2. Long incubation period 3. Model establishment success criteria are not established 4. The cultured model has an immature phenotype

Characteristics	Monolayer cell model	Stereo cell model	Organoid model
Advantages	1. Simple training 2. Model establishment time is short 3. Get results quickly 4. The model is easy to replicate	1. Maintain the material structure basis of the cellular microenvironment in the body 2. Some resistance to the growing environment 3. Cell-to-extracellular matrix interaction 4. The function of the cell can be expressed	1. Achieve similar natural organizational structure and function 2. Effectively reconstruct human tissue and have close physiology and pathophysiological status in vitro 3. Have some resistance to the growing environment 4. Has cell and extracellular matrix interactions 5. The function of the cell can be fully expressed
Disadvantages	1. Sensitive 2. High requirements for growth environment 3. Easy to be affected by many factors 4. Lack of cell and extracellular matrix interactions 5. Cell function cannot be fully expressed	1. Cultivation is more complicated 2. Model establishment time is longer 3. The result is slow 4. The model success rate is not high	1. Cultivate difficulties 2. Long incubation period 3. Model establishment success criteria are not established 4. The cultured model has an immature phenotype

Characteristics	Manual patch clamp	Automatic patch clamp	High-throughput cell imaging	Other molecular biology technique
Advantages	1. Highly recognized in the world 2. High sensitivity 3. Variety of test samples	1. Reduce technical requirements for personnel 2. Easier operation 3. High throughput 4. High efficiency 5. Stability	1. Large-scale research 2. Provide a variety of image analysis 3. Use a dedicated analysis approach to track multiple cellular processes 4. Observe the overall characteristics of the cell population 5. High throughput 6. Multiple indicators simultaneous detection 7. Saving samples 8. Detectable cells, tissue	1. Multi-technology combination, evaluation indicators increased 2. Mutual evidence to ensure accurate data
Disadvantages	1. Require professional technicians to operate 2. Low success rate 3. Long time 4. Low efficiency 5. Only detect electrical signal changes	1. Low sensitivity 2. The instrument is expensive 3. The detection object can only be a cell 4. Only detect changes in electrical signals	1. High requirements on the state and quality of the test object	1. Implementation is cumbersome 2. Long time 3. Waste of samples 4. Loss reagent

Characteristics	Manual patch clamp	Automatic patch clamp	High-throughput cell imaging	Other molecular biology technique
Advantages	1. Highly recognized in the world 2. High sensitivity 3. Variety of test samples	1. Reduce technical requirements for personnel 2. Easier operation 3. High throughput 4. High efficiency 5. Stability	1. Large-scale research 2. Provide a variety of image analysis 3. Use a dedicated analysis approach to track multiple cellular processes 4. Observe the overall characteristics of the cell population 5. High throughput 6. Multiple indicators simultaneous detection 7. Saving samples 8. Detectable cells, tissue	1. Multi-technology combination, evaluation indicators increased 2. Mutual evidence to ensure accurate data
Disadvantages	1. Require professional technicians to operate 2. Low success rate 3. Long time 4. Low efficiency 5. Only detect electrical signal changes	1. Low sensitivity 2. The instrument is expensive 3. The detection object can only be a cell 4. Only detect changes in electrical signals	1. High requirements on the state and quality of the test object	1. Implementation is cumbersome 2. Long time 3. Waste of samples 4. Loss reagent

类器官和立体细胞模型在中药心脏毒性评价中的应用前景

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刘昕彦 ¹^,²^,^# , 邵瑞 ¹^,²^,^# , 贺爽 ¹^,² , 朱彦 ¹^,²^,^*

药学学报 | 专题报道：中药心脏安全性评价及合理用于心血管疾病的防治 2019,54(11): 1888-1894

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药学学报 |专题报道：中药心脏安全性评价及合理用于心血管疾病的防治 2019 , 54 (11) : 1888 -1894

类器官和立体细胞模型在中药心脏毒性评价中的应用前景

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刘昕彦¹^,²^,^#, 邵瑞¹^,²^,^#, 贺爽¹^,², 朱彦¹^,²^,^*

作者信息

1.天津中医药大学, 天津市现代中药重点实验室, 天津 300193

2.天津国际生物医药联合研究院中药新药研发中心, 天津 300457

通讯作者:

*朱彦, Tel: 15822700439, E-mail: yanzhu.@icloud.com

Application prospect of organoids and 3D-cell models in evaluation of cardiotoxicity of traditional Chinese medicine

Xin-yan LIU¹^,², Rui SHAO¹^,², Shuang HE¹^,², Yan ZHU¹^,²^,^*

Affiliations

1. Tianjin Modern Chinese Medicine Key Laboratory, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China

2. Tianjin International Biomedical Research Institute, Chinese Medicine New Drug Research and Development Center, Tianjin 300457, China

出版时间: 2019-11-12 doi: 10.16438/j.0513-4870.2019-0105

文章导航

摘要

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心脏毒性是导致药物研发失败和上市后撤市的原因之一。然而，目前的药物心脏毒性评价方法存在临床相关性低、重现性低、处理量小等缺点。建立准确可靠的方法评价药物的心脏毒性是当前药物安全性评价及毒理学研究亟需解决的问题。心脏类器官作为新一代的药物心脏毒性评价模型，极大程度保留了心脏细胞在体内的生物特性和功能，更加真实、准确地反映药物对心脏的影响。本文就心脏立体细胞模型和类器官体外培养技术的进展作一综述，并重点讨论心脏类器官在中药心脏毒性评价中的应用及其发展潜力。文章还讨论了细胞和类器官模型在面对中药心脏毒性评价独特挑战方面的优势和前景。

关键词

中药 / 心脏毒性 / 二维细胞 / 三维细胞 / 类器官 / 毒性评价技术

Abstract

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Cardiotoxicity is one of the main causes of failure in new drug development or drug withdrawal from the market. However, current methods for evaluation of drug cardiotoxicity suffer the shortcomings such as low clinical relevance, low reproducibility and lack of high throughput screening capacity. Therefore, there is an urgent need for establishing more accurate and reliable methods for cardiotoxicity evaluation of drugs. As a new generation of drug cardiotoxicity evaluation, cardiac organs in culture retain the biological characteristics and functions of heart cells in the body, and can realistically and accurately respond to the effects of drugs. This article reviews recent progress of in vitro culture of cardiac organs and 3D-cell models, with focuses on application and development potential of cardiac organs for evaluation of cardiotoxicity of traditional Chinese medicine. The advantage and future prospective of such cell- and organ-based models for unique challenges in evaluation of cardiotoxicity of traditional Chinese medicine have been discussed.

Key words

traditional Chinese medicine / cardiotoxicity / two-dimensional cell / three-dimensional cell / organoid / toxicity evaluation technology

引用本文

刘昕彦, 邵瑞, 贺爽, 朱彦. 类器官和立体细胞模型在中药心脏毒性评价中的应用前景. 药学学报, 2019 , 54 (11) : 1888 -1894 . DOI: 10.16438/j.0513-4870.2019-0105

Xin-yan LIU, Rui SHAO, Shuang HE, Yan ZHU. Application prospect of organoids and 3D-cell models in evaluation of cardiotoxicity of traditional Chinese medicine[J]. Acta Pharmaceutica Sinica, 2019 , 54 (11) : 1888 -1894 . DOI: 10.16438/j.0513-4870.2019-0105

正文

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心脏是药物毒性的重要靶器官之一^[1]。传统心脏毒性评价通常使用动物模型, 通过检测给药后动物血清中乳酸脱氢酶、肌酸激酶同工酶等生化指标及心脏组织病理学的改变, 评价药物对实验动物心脏功能及组织形态的影响, 然而由于实验成本高^[2]、周期长、效率低、存在物种间差异及受伦理限制等问题, 阻碍了动物模型在药物筛选及评价中的广泛应用^[3]。近年来, 随着生命科学理论和技术的飞速发展, 心肌细胞体外心脏毒性评价模型因兼具成本低、操作简便等特点, 广泛应用于体外药物心脏毒性的评价和有毒成分的筛选。其中, 采用hERG稳定转染的HEK293细胞系评价药物对心脏电生理特性的影响, 是体外研究药物心脏毒性细胞模型的金标准^[4]。而心脏立体细胞培养技术是近年来新兴的一种用于体外药物心脏毒性评价的细胞培养技术, 能够最大限度地在体外模拟体内的组织结构和功能, 可以更加高效、准确评估药物引起的心脏毒副作用。

中草药作为一种宝贵且天然的资源, 因其来源广、价格低、多靶点、多调控、疗效好和不良反应小已被广泛应用于各种疾病的治疗^[5]。我国上市的中药大部分疗效确切安全方便, 但仍有部分药物存在一定的毒副作用和安全隐患^[6]。近年中药引起的不良反应文献报道日趋增加, 尤其是活血、强心类中药引起心脏方面的毒性报道不在少数^[7-9], 此外, 一些非心血管类药物如常用于治疗风湿关节炎、痛风疾病的雷公藤、关木通和汉防己等中药也被报道可影响心脏功能, 具有心脏毒性^[10-12]。如何在新药研发和临床前较为详细和全面地评价药物心脏安全性, 降低中药心脏毒性风险, 是目前必须正视和重视的问题。在Pubmed数据库、中国知网数据库、万方和维普数据库中以关键词心脏毒性、类组织、中药、三维(3D)细胞交叉搜索不足千篇。本文总结了心脏类器官及心脏立体细胞培养技术的最新研究进展, 进一步结合体外中药心脏毒性评价模型的研究现状, 对类器官和立体细胞模型应用于中药心脏毒性评价的前景进行展望。

1 心脏立体细胞模型

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立体细胞模型是细胞在类似体内生长环境的支架或基质中, 通过紧密连接或缝隙连接等连接方式建立细胞间及细胞与胞外基质间的联系, 并形成一定的三维结构。目前, 关于心脏立体细胞培养模型的研究已取得了一定的进展。Correia等^[13]采用诱导多能干细胞分化产生的心肌细胞(hiPSC-CM), 在搅拌条件下培养形成高纯度的心肌细胞聚集体, 进一步研究发现, 与平面培养的心肌细胞相比, 参与糖酵解和脂质生物合成的基因显著下调, 而参与氧化磷酸化的基因表达明显增加, 细胞间相互作用及胞外基质对基因的表达影响明显, 提示立体模型的使用相较于平面模型考评指标更有参考价值。Trac等^[14]基于心脏祖细胞(CPC), 将其聚集成无支架球体, 显著增加Notch1的基因表达, 相比平面CPC培养, 心功能得到改善, CPC立体模型移植可能作为一种潜在的小儿心力衰竭治疗方法, 但仍需要进一步研究。Yue等^[15]在体外诱导合成了包含心肌细胞(CMs)和人类诱导多能干细胞(hiPSC)衍生内皮细胞(iECs)的仿生合成心肌组织, 并在此基础上建立心肌梗死模型, 与诱导二维(2D)细胞培养相比, 细胞存活率有所提高。更重要的是, 该基因表达谱在共培养CMs中对氧化应激反应保持不变, 表明iECs在氧化应激条件下对CMs具有稳定作用。并得到iECs通过稳定线粒体复合物、抑制氧化磷酸化通路、激活心肌细胞代谢-细胞色素P450通路、Rap1信号通路和肾上腺素能信号通路等途径, 对氧化应激下CMs具有保护作用的结论。采用此模型对治疗心肌梗死的药物进行毒性评价, 更贴近人体内环境, 实验结果将具有更高的临床价值。Campbell等^[16]开发了一种新型的体外人体心脏三维模型, 称之为血管化心脏球体(VCS)。主要通过将诱导hiPSC产生的心肌细胞、心脏成纤维细胞和人类冠状动脉内皮细胞, 以类似于人体内心脏的比例在悬滴培养模型中共同培养。可观察到三维细胞组织、细胞外基质和微血管网络的形成, 极大程度模拟了体内的心脏组织。

目前, 部分心脏立体细胞培养模型已应用于化疗药物的心脏毒性评价研究中。Varzideh等^[17]使用了人体3D心脏微组织(InSphero, SWL)模型即体外球体细胞模型中包含约4 000个人类心肌细胞和1 000个心脏成纤维细胞联合药代动力学模型, 模拟了特定剂量的多柔比星(DOX)在人体中的吸收、分布、代谢和排泄, 成功检测DOX 3种主要毒性过程中的生物变化(心肌细胞功能障碍、线粒体功能障碍和细胞死亡)。心肌细胞与心肌成纤维细胞共培养, 优化了组织结构, 增强了心脏微组织的结构和功能特性。

2 心脏类器官

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类器官最早是在1946年被Smith和Cochrae提出并用于描述1例囊性畸胎瘤^[18]。而如今类器官用于在体外重建人体组织, 并模拟生理结构和功能以及准确地呈现病理生理状况。类器官虽提出较早, 却受条件所限, 而近年生物材料和3D打印技术等方面取得的重大突破, 推动了类器官研究的飞速发展, 尤其是在肿瘤^[19-26]、消化^{[27, 28]}、神经^[29-32]、内分泌和心血管等医学领域中的模型建立及应用的探索引起广泛关注。目前在体外心脏模型中类器官的构建较为复杂, 除了尽可能模仿体内生理状态, 空间上的排列也成为模型优化的一个考虑因素。Lemme等^[33]将hiPSC产生的CM建立右心房工程心脏组织(RA-EHT)作为人心房的3D模型, 与人的肌肉比较, RA-EHT表现出更高的mRNA和蛋白质浓度的心房指标, 更快的收缩动力学, 更低的力生成、更短的动作电位持续时间和更高的复极化分数, 真实反映了心房心室肌差异, 提示RA-EHT可作为人类心房的模型, 用于临床前药物筛选。Rogozhnikov等^[34]利用生物正交化学和细胞表面工程相结合的方法, 采用心肌细胞、心肌成纤维细胞和人脐静脉内皮细胞(HUVECs) 3种不同类型的细胞快速自组装成具有功能的3D心脏组织, 通过酮或含氧胺的脂质体处理(脂质体融合过程在几秒到几分钟内完成), 并将生物正交基团安装到细胞表面(脂质体融合/生物正交传递技术适用于许多哺乳动物细胞类型, 而且速度快、温和, 在几秒钟内就能将生物正交基团安装到刚从大鼠心脏中提取的心肌细胞表面), 评估了异丙肾上腺素和DOX对不同心脏组织的心率影响。这些结果表明, 无支架的3D心脏组织在没有外界刺激的情况下自发搏动, 并可对已知的心肌细胞药物刺激产生相应的反应。Zhang等^[35]通过3D生物打印技术生成多层水凝胶微纤维支架包裹内皮细胞, 形成管腔状结构和血管床并接种上心肌细胞形成内皮化心肌, 将其嵌入到专门设计的微流体灌注生物反应器, 通过证明细胞导电、肌节存在产生心肌搏动基础, 可以完成内皮细胞-芯片心肌平台的心血管毒性评估。Hoang等^[36]基于生物材料的细胞模式与干细胞类器官工程相结合, 将2D人类诱导多能干细胞集落产生3D心脏微腔。这些微小的近似于早期发展的心脏, 具有独特的空间组织和自我组装, 然后经过光刻微加工, 可以作为发现新药的体外模型, 以及调节胎儿发育的处方药经受胚胎毒性筛选实验时的体外模型。Rogers等^[37]设计了一种仿生心脏组织芯片(CTC)模型, 其中在3D纤维中包裹的H9c2细胞经受血液动力影响, 模拟左心室中压力-体积变化, 可以精确控制与心率相关的各种参数如心率、收缩期峰值压力、舒张末期压力和体积、收缩末期压力和体积以及收缩压和舒张压之间的持续时间比可用于相关疾病模型研究。Lee团队^[38]提出了由人胚胎干细胞(hESC)衍生的CM间充质干细胞(MSCs)组成的收缩性心肌微组织的3D心脏球体平台, 通过转化生长因子-β1 (TGF-β1)诱导心肌纤维化, 建造体外心脏纤维化模型模拟体内心脏复杂的病理环境, 表明3D心脏微组织具有显示和评估各种药物的促纤维化作用的潜力, 这对在体外预测药物诱导的心脏纤维化和研究人类心肌纤维化的病理变化提供了很好的研究方法。Yang等^[39]通过结合3D氧化铁支架(IOS)与固定比例的人多能干细胞(hPSC)来源的心室特异性心肌细胞和人类脐带间充质干细胞, 构建了一种新型的人类心室特异性心脏组织(EhVHT), EhVHT促进心脏特异性基因的表达, 离子交换, 并在体外表现出更好的钙离子行为和正常的电生理活性, EhVHT有效地促进了体内心脏组织的修复, 并促进了急性心肌梗死(MI)大鼠受损心脏功能的恢复, 可以满足大多数MI病例心室损伤模型的要求, 以及可用于筛查特异性靶向心室心肌的药物。

从单细胞球体模型发展到两种细胞共培养的3D球体模型再到多种细胞互作的3D微组织模型以及有支架的多种细胞共培养的类心脏器官模型, 技术上更新迅速, 模型更为复杂, 研究人员对心脏这一组成复杂的器官研究更为细致和具体, 已经有意识地构造心脏相关的腔室模型、细化分区及努力构造相应疾病模型, 类器官相比立体细胞模型, 借助了生物工程技术, 在细胞间相互作用和分泌的物质以及微环境更贴近心脏的生理状态, 也表现出比单细胞培养更成熟表型, 对药物作用评价更准确(表 1)。

3 体外中药心脏毒性筛选的相关应用

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随着中药在临床上使用量的日益增加, 准确、合理和高通量的有毒中药筛选需求也日益增强, 虽然目前体外中药心脏毒性的评价主要采用的仍是单层心肌细胞培养, 但膜片钳技术^[40-42]、高内涵影像分析技术、核磁和气质联用的代谢组学等实验技术的相继提升, 大大缩减了基于单层细胞培养模型的体外中药心脏毒性评价的时间和成本, 推动了中药体外心脏毒性评价方法的发展。

3.1 全自动膜片钳技术

膜片钳是一种以记录通过离子通道的离子电流来反映细胞膜上单一或多数的离子通道分子活动的技术。该技术可应用于许多细胞系的研究, 也是目前唯一可记录1个蛋白分子电活动的方法。在过去的10年, 全自动膜片钳技术逐渐发展起来, 除了能够记录异源表达的电流, 还能够记录内源性的电流, 如胚胎干细胞(ESC)或iPSC-CM中的电流^{[43, 44]}。心肌电生理功能变化如动作电位离子通道电流异常经常与心律失常、心衰和心肌缺血等心血管疾病联系密切。因此, 心脏电生理功能指标是体外中药心脏毒性评价需考察的首要指标。如Chen^[45]记录了雷公藤红素对原代心肌细胞动作电位的影响, 通过电生理揭示了雷公藤红素抑制原代心肌细胞的离子通道和破坏正常电信号的潜在机制。Wang等^[46]建立基于全自动膜片钳平台的中药体外心脏毒性评价方法, 并对1 036个中草药提取物进行心脏毒性筛选和评价, 进一步证明集成的高通量hERG膜片钳和高含量多参数成像心脏毒性筛选方法可用于复杂中草药的大规模临床前评估。Becker等^[43]将记录iPSC-CM电流的实验方法进行优化, 证明膜片钳技术可用于iPSC-CM细胞检测。Scheel等^[47]也验证了hiPSC-CM电生理特性。说明用全自动膜片钳通过检测离子电流而开展心脏毒性评价逐步成熟。

手动膜片钳本身对于细胞膜电信号的检测在世界范围内得到广泛认可, 可通过对离子通道的抑制和电信号的破坏作为指标检测药物对细胞影响, 可以考虑作为心脏毒性评价的一个指标; 然而, 手动膜片钳受技术手法限制, 要求专业素养高的技术人员操作, 成功率低且耗时长、效率低, 可能更适用于化学药单体的研究, 在中药这种多成分的复杂组合中一个一个去实验是不切合实际的。全自动膜片钳基于手动膜片钳的工作原理, 在对人员技术要求降低的同时, 使操作更简便、高通量, 提高效率和稳定性, 可同时对384个组分或单体进行检测。Wu等^[48]展示了一种新的、用于急性脑切片的体外全细胞膜片钳实验的图像引导自动膜片钳系统, 可以应用于解离神经元、器官型切片培养和其他非神经元细胞类型, 扩大了研究对象和样本类型, 为中药研究拓宽了研究方向。

3.2 高内涵细胞影像分析技术

高内涵细胞影像技术是以细胞为单位, 通过荧光标记物在高分辨率的荧光显微镜下高速、灵敏地获得细胞生长过程中受外因作用改变或未改变的相关图像及量化后的参数。Zhu等^[49]采用H9c2大鼠心肌细胞系, 建立并优化了基于细胞影像的高内涵多指标评价中药心脏毒性的方法。Zhang^[50]采用类似方法检测了丹参6个馏分对DOX诱导的H9c2心肌细胞损伤过程中的保护作用时, 发现丹参6个馏分中编号06馏分有显著性细胞毒性。Ren^[51]检测麦冬中各成分对H9c2细胞的毒性作用时, 发现麦冬皂苷D'可致心肌细胞损伤。Cui等^[52]采用高内涵筛选和流式细胞术结合溴脱氧尿苷(Brd U)标记方法对心肌成纤维细胞(CFs)增殖和周期进行分析。Zhang等^[53]采用实时细胞分析系统和高内涵分析技术, 监测黄连(RC)中9种生物碱在CMs治疗中的作用; 采用荧光酶偶联三磷酸腺苷(ATP)检测细胞活力, 首次对9种生物碱的心脏毒性进行了评价, 阐明RC中的心脏毒性成分。

高内涵细胞影像分析技术的出现, 大大提高了“有毒”中药的筛选效率。Chen等^[54]通过高通量虚拟筛选1 000万个化合物及基于细胞的验证, 发现了一种新的非脂肽样化合物, 并评估该物质对Toll样受体的先天免疫反应。Halaidych等^[55]对于不同起源的血管平滑肌细胞采用高通量细胞成像技术跟踪单个细胞分析Ca²⁺释放动力学和收缩, 可同时对数百个细胞进行分析, 而手动低通量检测或标记物可能会忽略对血管收缩剂反应的异质性表达。

基于中药多组分、多靶点和多通路的特点, 目前人们已将更多目光集中在如何快速、高通量和多指标地综合性评价中药心脏毒性, 联合高通量技术及其他分子技术, 全面而又系统的中药药物心脏毒性评价方法必将成为研究主流手段。高通量技术的出现, 大大缩减了基于2D细胞培养模型的体外中药心脏毒性评价的时间和成本, 推动了中药体外心脏毒性评价方法的发展, 低效、低通量的筛选技术注定会被取代(表 2)。

4 总结与展望

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目前单层细胞培养模型在中药毒性的评价上应用比较广泛, 但对环境要求较高, 容易受外界因素影响。此外, 心肌细胞在2D的培养环境中缺乏细胞和细胞外基质相互作用, 不具有心脏的生理功能, 会影响剂量有效性和剂量毒性的预测, 并阻碍细胞的功能完整表达, 在药物安全性评价中具有一定的局限性, 对于中药的多组分多靶点的作用机制难以展示整体作用, 投入的时间和精力往往是化学药研究的百倍至千倍, 大大增加了中药研究工作者的研究难度, 也成为中药新药开发的一大难题。三维立体细胞培养既能保留体内细胞微环境的物质结构基础, 又能体现细胞培养的直观性及条件可控制性, 把体外无细胞及单层细胞培养体系与组织器官及整体研究联系起来, 是目前研究的新兴热点。三维细胞培养模型也广泛用于治疗心血管疾病的西药的心脏毒性评价研究中^[56-58]。类器官研究已经成为各热门医疗领域研究的热点, 通过改进细胞的体外培养条件, 在体外再现体内细胞微环境, 最终诱导生成具有类似生理功能的类组织, 模拟在体环境或组织环境来评价药物, 使得药物作用引起的变化更贴近机体反应。尽管目前培养技术还未大量应用于中药心脏毒性评价的研究, 但是其真实高效的特点是2D细胞培养和动物模型所不具备的。类器官的研究取得了一定的进展, 可从空间和生理功能方面模拟体内器官, 但仍处于起步阶段, 在药物毒性评价方面尚未出现过多报道, 然而需要注意的是, 三维组织培养向类器官进化过程艰难且具有挑战性, 体外立体细胞培养虽然大体上接近于活体水平组织的特征, 但仍存在一些问题: ①技术层面上, 类器官及立体细胞培养是否成功, 评价标准需要建立; ②培养的微环境需要进一步优化, 营造类似体内微环境; ③新培养的类器官或立体细胞模型在功能上可能与成人心脏有所差异, 可能其状态如同新生儿一样功能发育不完全, 心肌细胞的不成熟表型是其在转化医学、体外药物毒性和药理分析中的重要障碍。如何诱导发育成熟也是完善模型另一个考虑的因素。随着多种新模型出现, 培养的时间和投入的精力较之以前更多, 这使得研究过程速度更为缓慢, 若能与高通量新技术相互结合应用于中药心脏毒性研究, 将带动该领域的快速发展, 技术服务于研究鉴于类心脏组织在化学药中毒性评价应用, 利用该体外模型评价中药的心脏毒性结合高通量技术是可行的。

基金

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国家重点研发计划项目(2018YFC1704500)
国家科技重大专项(2018ZX01031301)
国家自然科学基金资助项目(81873037)

参考文献

收起

文献

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参考文献引证文献

排序方式：

[1]

Pereira

, Silva

, Diogo

et al . Drug-induced cardiac mitochondrial toxicity and protection: from doxorubicin to carvedilol[J]. Curr Pharm Des, 2011, 17: 2113-2129.

https://doi.org/10.2174/138161211796904812

[2]

Liang

, Li

. A novel network pharmacology approach to analyse traditional herbal formulae: the Liu-Wei-Di-Huang pill as a case study[J]. Mol Biosyst, 2014, 10: 1014-1022.

https://doi.org/10.1039/C3MB70507B

[3]

Tan

, Ko

, Liu

et al . Serum metabolomics reveals betaine and phosphatidylcholine as potential biomarkers for the toxic responses of processed Aconitum carmichaelii Debx[J]. Mol Biosyst, 2014, 10: 2305-2316.

https://doi.org/10.1039/C4MB00072B

[4]

Liang

, Lan

, Lee

et al . Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity[J]. Circulation, 2013, 127: 1677-1691.

https://doi.org/10.1161/CIRCULATIONAHA.113.001883

[5]

, You

, Li

et al . MicroRNA-542-3p suppresses tumor cell proliferation via targeting Smad2 inhuman osteosarcoma[J]. Oncol Lett, 2018, 15: 6895-6902. http://www.researchgate.net/publication/323707129_MicroRNA-542-3p_suppresses_tumor_cell_proliferation_via_targeting_Smad2_inhuman_osteosarcoma

[6]

. Adverse reactions and preventive measures of oral chinese medicine[J]. China Health Stand Manag (中国卫生标准管理), 2017, 8: 92-93. http://en.cnki.com.cn/Article_en/CJFDTOTAL-WSBZ201725047.htm

[7]

Gui

, Chen

, Lin

et al . Cytotoxicity effects of shikonin on rat cardiomyocytes in vitro[J]. Pharmacol Clin Chin Mater Med (中药药理与临床), 2010, 26: 33-36. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zyylylc201006013

[8]

, Wang

, Ouyang

et al . Influences of the Toad Venom on Bufo gargarizans heart activities[J]. J Jilin Norm Univ (Nat Sci Ed) (吉林师范大学学报(自然科学版)), 2014, 35: 142-144. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=slxk-zrkx201403035

[9]

Huang

, Liu

, Meng

et al . Effect of nitidine chloride on the heart development of zebrafish embryos[J]. Guangxi Med J (广西医学), 2011, 33: 546-548. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=gxyx201105011

[10]

Wang XC. Comparsion of the Arrhythmogenic Effects between ACO and MACO on Ventricular Myocytes of Guinea-pig and Their Underlying Cellular Mechanisms (乌头碱和新乌头碱致心律失常作用比较及其细胞学机制)[D]. Shijiazhuang: Hebei Medical University, 2013. http://cdmd.cnki.com.cn/Article/CDMD-10089-1013287871.htm

[11]

Huang

, Chen

, Huang

et al . Aristolochic acid induces heart failure in zebrafish embryos that is mediated by inflammation[J]. Toxicol Sci, 2007, 100: 486-494.

https://doi.org/10.1093/toxsci/kfm235

[12]

Wang

, Liu

, Wang

et al . Preliminary study on cardiotoxicity of celastrol to zebrafish embryo[J]. Chin Pharmacol Bull (中国药理学通报), 2009, 25: 634-636. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zgylxtb200905019

[13]

Correia

, Koshkin

, Duarte

et al . 3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes[J]. Biotechnol Bioeng, 2018, 115: 630-644.

https://doi.org/10.1002/bit.26504

[14]

Trac

, Maxwell

, Brown

et al . Aggregation of child cardiac progenitor cells into spheres activates notch signaling and improves treatment of right ventricular heart failure[J]. Circ Res, 2019, 124: 526-538.

https://doi.org/10.1161/CIRCRESAHA.118.313845

[15]

Yue

, Acun

, Zorlutuna

. Transcriptome profiling of 3D co-cultured cardiomyocytes and endothelial cells under oxidative stress using a photocrosslinkable hydrogel system[J]. Acta Biomater, 2017. DOI:10.1016/j.actbio.2017.06.031.

https://doi.org/10.1016/j.actbio.2017.06.031

[16]

Campbell

, Chabria

, Figtree

et al . Stem cell-derived cardiac spheroids as 3D in vitro models of the human heart microenvironment[J]. Methods Mol Biol, 2018. DOI:10.1007/7651_2018_187.

https://doi.org/10.1007/7651_2018_187

[17]

Varzideh

, Pahlavan

, Ansari

et al . Human cardiomyocytes undergo enhanced maturation in embryonic stem cell-derived organoid transplants[J]. Biomaterials, 2019. DOI:10.1016/j.biomaterials.2018.11.033.

https://doi.org/10.1016/j.biomaterials.2018.11.033

[18]

Smith

, Cochrane

. Cystic organoid teratoma: (report of a case)[J]. Can Med Assoc J, 1946, 55: 151-152. http://d.old.wanfangdata.com.cn/OAPaper/oai_pubmedcentral.nih.gov_1582935

[19]

Weeber

, Ooft

, Dijkstra

et al . Tumor organoids as a pre-clinical cancer model for drug discovery[J]. Cell Chem Biol, 2017, 24: 1092-1100.

https://doi.org/10.1016/j.chembiol.2017.06.012

[20]

Cortina

, Turon

, Stork

et al . A genome editing approach to study cancer stem cells in human tumors[J]. EMBO Mol Med, 2017, 9: 869-879.

https://doi.org/10.15252/emmm.201707550

[21]

Briem

, Ingthorsson

, Traustadottir

et al . Application of the D492 cell lines to explore breast morphogenesis, EMT and cancer progression in 3D culture[J]. Mammary Gland Biol Neoplasia, 2019. DOI:10.1007/s10911-018-09424-w.

https://doi.org/10.1007/s10911-018-09424-w

[22]

Kar

, Molla

, Katti

et al . Tissue-engineered nanoclay-based 3D in vitro breast cancer model for studying breast cancer metastasis to bone[J]. J Tissue Eng Regen Med, 2019, 13: 119-130.

https://doi.org/10.1002/term.2773

[23]

Zhao

, Yan

, Hu

et al . Sphere‑forming assay vs organoid culture: determining long‑term stemness and the chemoresistant capacity of primary colorectal cancer cells[J]. Int J Oncol, 2019, 54: 893-904.

[24]

Fontana

, Raimondi

, Marzagalli

et al . Epithelial-to-mesenchymal transition markers and CD44 isoforms are differently expressed in 2D and 3D cell cultures of prostate cancer cells[J]. Cells, 2019. DOI:10.3390/cells8020143.

https://doi.org/10.3390/cells8020143

[25]

Haq

, Samuel

, Haxho

et al . Sialylation facilitates self-assembly of 3D multicellular prostaspheres by using cyclo-RGDfK (TPP) peptide[J]. Onco Targets Ther, 2017, 10: 2427-2447.

https://doi.org/10.2147/OTT.S133563

[26]

Guan

, Xu

, Garfin

et al . Human hepatic organoids for the analysis of human genetic diseases[J]. JCI Insight, 2017, 2: 94954.

https://doi.org/10.1172/jci.insight.94954

[27]

Anabazhagan

, Chatterjee

, Priyamvada

et al . Methods to study epithelial transport protein function and expression in native intestine and caco-2 cells grown in 3D[J]. J Vis Exp, 2017. DOI:10.3791/55304.

https://doi.org/10.3791/55304

[28]

Fernando

, Dicay

, Stahl

et al . A simple, cost-effective method for generating murine colonic 3D enteroids and 2D monolayers for studies of primary epithelial cell function[J]. Am J Physiol Gastrointest Liver Physiol, 2017, 313: G467-G475.

https://doi.org/10.1152/ajpgi.00152.2017

[29]

Kaisar

, Sajja

, Prasad

et al . New experimental models of the blood-brain barrier for CNS drug discovery[J]. Expert Opin Drug Discov, 2017, 12: 89-103.

https://doi.org/10.1080/17460441.2017.1253676

[30]

Pamies

, Barreras

, Block

et al . A human brain microphysiological system derived from induced pluripotent stem cells to study neurological diseases and toxicity[J]. ALTEX, 2017, 34: 362-376. http://www.ncbi.nlm.nih.gov/pubmed/27883356

[31]

Fan

, Liu

, Chen

et al . Directing induced pluripotent stem cell derived neural stem cell fate with a three-dimensional biomimetic hydrogel for spinal cord injury repair[J]. ACS Appl Mater Interfaces, 2018, 10: 17742-17755.

https://doi.org/10.1021/acsami.8b05293

[32]

Cai

, Chen

, Zhou

et al . Research advancement in the construction and applications of microfluidic devices for in vitro blood-brain barrier research[J]. Acta Pharm Sin (药学学报), 2019, 54: 269-280. http://www.yxxb.com.cn:8081/aps/CN/abstract/abstract17414.shtml

[33]

Lemme

, Ulmer

, Lemoine

et al . Atrial-like engineered heart tissue: an in vitro model of the human atrium[J]. Stem Cell Rep, 2018, 11: 1378-1390.

https://doi.org/10.1016/j.stemcr.2018.10.008

[34]

Rogozhnikov

, O'Brien

, Elahipanah

et al . Scaffold free bio-orthogonal assembly of 3-dimensional cardiac tissue via cell surface engineering[J]. Sci Rep, 2016, 6: 39806.

https://doi.org/10.1038/srep39806

[35]

Zhang

, Arneri

, Bersini

et al . Bioprinting 3D microfibrous scaffolds for engineering endothelialized myocardium and heart-on-a-chip[J]. Biomaterials, 2016. DOI:10.1016/j.biomaterials.2016.09.003.

https://doi.org/10.1016/j.biomaterials.2016.09.003

[36]

Hoang

, Wang

, Conklin

et al . Generation of spatial-patterned early-developing cardiac organoids using human pluripotent stem cells[J]. Nat Protoc, 2018, 13: 723-737.

https://doi.org/10.1038/nprot.2018.006

[37]

Rogers

, Miller

, Kannappan

et al . Cardiac tissue chips (CTCs) for modelling cardiovascular disease[J]. IEEE Trans Biomed Eng, 2019. DOI:10.1109/TBME.2019.2905763.

https://doi.org/10.1109/TBME.2019.2905763

[38]

Lee

, Jung

, Jo

et al . Modelling cardiac fibrosis using three-dimensional cardiac microtissues derived from human embryonic stem cells[J]. J Biol Eng, 2019. DOI:10.1186/s13036-019-0139-6.

https://doi.org/10.1186/s13036-019-0139-6

[39]

Yang

, Wei

, Liu

et al . Engineering human ventricular heart tissue based on macroporous iron oxide scaffolds[J]. Acta Biomater, 2019. DOI:10.1016/j.actbio.2019.02.024.

https://doi.org/10.1016/j.actbio.2019.02.024

[40]

Gao

, Wang

, Song

et al . Progress in electrophysiological research of cardiomyocytes in traditional Chinese medicine[J]. Chin J Integr Med Cardio (中西医结合心脑血管病杂志), 2016, 14: 35-38.

[41]

Cao

, Zheng

, Lu

et al . Advancement in ion channel research-automation patch clamp technology[J]. Mod Instrument (现代仪器), 2007, 13: 47-50. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=xdyq200702016

[42]

Chen

, Zhu

. Application potentials of hiPSC-derived cardiomyocytes in preclinical cardiotoxicity screening and post-marketing safety reevaluation of Chinese medicine[J]. Tianjin J Tradit Chin Med (天津中医药), 2017, 34: 76-81. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=tjzy201702003

[43]

Becker

, Stoelzle

, G pel

et al . Minimized cell usage for stem cell-derived and primary cells on an automated patch clamp system[J]. J Pharmacol Toxicol Methods, 2013, 68: 82-87.

https://doi.org/10.1016/j.vascn.2013.03.009

[44]

Stoelzle

, Haythornthwaite

, Kettenhofen

et al . Automated patch clamp on m ESC-derived cardiomyocytes for cardiotoxicity prediction[J]. J Biomol Screen, 2011, 16: 910-916.

https://doi.org/10.1177/1087057111413924

[45]

Chen Z. Molecular Mechanism of Cardiotoxicity Induced by Tripterine (雷公藤红素致心脏毒性的分子机制研究)[D]. Nanjing: Nanjing Normal University, 2012. http://cdmd.cnki.com.cn/Article/CDMD-10319-1015569819.htm

[46]

Wang

, Chen

, Yu

et al . High-throughput electrophysiology screen revealed cardiotoxicity of strychnine by selectively targeting hERG channel[J]. Am J Chin Med, 2018, 46: 1825-1840.

https://doi.org/10.1142/S0192415X1850091X

[47]

Scheel

, Frech

, Amuzescu

et al . Action potential characterization of human induced pluripotent stem cell-derived cardiomyocytes using automated patch-clamp technology[J]. Assay Drug Dev Technol, 2014, 12: 457-469.

https://doi.org/10.1089/adt.2014.601

[48]

, Chubykin

. Application of automated image-guided patch clamp for the study of neurons in brain slices[J]. J Vis Exp, 2017. DOI:10.3791/56010.

https://doi.org/10.3791/56010

[49]

Zhu

, Wang

, Zhu

. Quantitative cardiotoxicity assessment of gambogic acid using multiple cellular phenotype analysis[J]. Chin J Pharmacol Toxicol (中国药理学与毒理学杂志), 2017, 31: 73-79. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zgylxydl201701009

[50]

Zhang Q. The Pharmacological Components and Mechanism of Salvia Miltiorrhiza Were Found Based on the Myocardial Injury Model Induced by Doxorubicin (基于多柔比星致心肌损伤模型发现丹参药效成分及作用机制)[D]. Beijing: Beijing University of Chinese Medicine, 2017. http://cdmd.cnki.com.cn/Article/CDMD-10026-1017162251.htm

[51]

Ren SJ. Discovery of Cardiotoxicity Caused by Ophiopogon Saponins D' and Its Mechanism (麦冬皂苷D'致心脏毒性的发现及其机制研究)[D]. Nanning: Guangxi Medical University, 2018. http://cdmd.cnki.com.cn/Article/CDMD-10598-1018156399.htm

[52]

Cui

, Li

, Wu

et al . Application of high-content screening and flow cytometry analysis techniques to evaluation of myocardial fibroblasts proliferation[J]. Acta Physiol Sin (生理学报), 2014, 66: 215-222. http://cn.bing.com/academic/profile?id=67f31131b803e6ed40c82b2020bd080a&encoded=0&v=paper_preview&mkt=zh-cn

[53]

Zhang

, Yu

, Wang

et al . Cardiotoxicity evaluation of nine alkaloids from Rhizoma Coptis[J]. Hum Exp Toxicol, 2018, 37: 185-195.

https://doi.org/10.1177/0960327117695633

[54]

Chen

, Cen

, Yang

et al . Synthesis of urea analogues bearing N-alkyl-N'-(thiophen-2-yl) scaffold and evaluation of their innate immune response to toll-like receptors[J]. Eur J Med Chem, 2019. DOI:10.1016/j.ejmech.2019.02.067.

https://doi.org/10.1016/j.ejmech.2019.02.067

[55]

Halaidych

, Cochrane

, van den Hil

et al . Quantitative analysis of intracellular Ca²⁺ release and contraction in hiPSC-derived vascular smooth muscle cells[J]. Stem Cell Rep, 2019, 12: 647-656.

https://doi.org/10.1016/j.stemcr.2019.02.003

[56]

Archer

, Sargeant

, Basak

et al . Characterization and validation of a human 3D cardiac microtissue for the assessment of changes in cardiac pathology[J]. Sci Rep, 2018, 8: 10160.

https://doi.org/10.1038/s41598-018-28393-y

[57]

Takeda

, Miyagawa

, Fukushima

et al . Development of in vitro drug-induced cardiotoxicity assay by using three-dimensional cardiac tissues derived from human induced pluripotent stem cells[J]. Tissue Eng Part C Methods, 2018, 24: 56-67.

https://doi.org/10.1089/ten.tec.2017.0247

[58]

, Leong

, Lim

et al . Engineering a functional three-dimensional human cardiac tissue model for drug toxicity screening[J]. Biofabrication, 2017, 9: 025011.

https://doi.org/10.1088/1758-5090/aa6c3a

2019年第54卷第11期

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文章信息

doi: 10.16438/j.0513-4870.2019-0105

接收时间：2019-02-14
首发时间：2026-01-26
出版时间：2019-11-12

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文章信息

作者

出版历史

收稿日期：2019-02-14
修回日期：2019-03-28

基金

国家重点研发计划项目(2018YFC1704500)

国家科技重大专项(2018ZX01031301)

国家自然科学基金资助项目(81873037)

作者信息

1.天津中医药大学, 天津市现代中药重点实验室, 天津 300193

2.天津国际生物医药联合研究院中药新药研发中心, 天津 300457

通讯作者:

*朱彦, Tel: 15822700439, E-mail: yanzhu.@icloud.com

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Characteristics	Monolayer cell model	Stereo cell model	Organoid model
Advantages	1. Simple training 2. Model establishment time is short 3. Get results quickly 4. The model is easy to replicate	1. Maintain the material structure basis of the cellular microenvironment in the body 2. Some resistance to the growing environment 3. Cell-to-extracellular matrix interaction 4. The function of the cell can be expressed	1. Achieve similar natural organizational structure and function 2. Effectively reconstruct human tissue and have close physiology and pathophysiological status in vitro 3. Have some resistance to the growing environment 4. Has cell and extracellular matrix interactions 5. The function of the cell can be fully expressed
Disadvantages	1. Sensitive 2. High requirements for growth environment 3. Easy to be affected by many factors 4. Lack of cell and extracellular matrix interactions 5. Cell function cannot be fully expressed	1. Cultivation is more complicated 2. Model establishment time is longer 3. The result is slow 4. The model success rate is not high	1. Cultivate difficulties 2. Long incubation period 3. Model establishment success criteria are not established 4. The cultured model has an immature phenotype

Characteristics

Monolayer cell model

Stereo cell model

Organoid model

Advantages

1. Simple training
2. Model establishment time is short
3. Get results quickly
4. The model is easy to replicate

1. Maintain the material structure basis of the cellular microenvironment in the body
2. Some resistance to the growing environment
3. Cell-to-extracellular matrix interaction
4. The function of the cell can be expressed

1. Achieve similar natural organizational structure and function
2. Effectively reconstruct human tissue and have close physiology and pathophysiological status in vitro
3. Have some resistance to the growing environment
4. Has cell and extracellular matrix interactions
5. The function of the cell can be fully expressed

Disadvantages

1. Sensitive
2. High requirements for growth environment
3. Easy to be affected by many factors
4. Lack of cell and extracellular matrix interactions
5. Cell function cannot be fully expressed

1. Cultivation is more complicated
2. Model establishment time is longer
3. The result is slow
4. The model success rate is not high

1. Cultivate difficulties
2. Long incubation period
3. Model establishment success criteria are not established
4. The cultured model has an immature phenotype

Characteristics	Manual patch clamp	Automatic patch clamp	High-throughput cell imaging	Other molecular biology technique
Advantages	1. Highly recognized in the world 2. High sensitivity 3. Variety of test samples	1. Reduce technical requirements for personnel 2. Easier operation 3. High throughput 4. High efficiency 5. Stability	1. Large-scale research 2. Provide a variety of image analysis 3. Use a dedicated analysis approach to track multiple cellular processes 4. Observe the overall characteristics of the cell population 5. High throughput 6. Multiple indicators simultaneous detection 7. Saving samples 8. Detectable cells, tissue	1. Multi-technology combination, evaluation indicators increased 2. Mutual evidence to ensure accurate data
Disadvantages	1. Require professional technicians to operate 2. Low success rate 3. Long time 4. Low efficiency 5. Only detect electrical signal changes	1. Low sensitivity 2. The instrument is expensive 3. The detection object can only be a cell 4. Only detect changes in electrical signals	1. High requirements on the state and quality of the test object	1. Implementation is cumbersome 2. Long time 3. Waste of samples 4. Loss reagent

Characteristics

Manual patch clamp

Automatic patch clamp

High-throughput cell imaging

Other molecular biology technique

Advantages

1. Highly recognized in the world
2. High sensitivity
3. Variety of test samples

1. Reduce technical requirements for personnel
2. Easier operation
3. High throughput
4. High efficiency
5. Stability

1. Large-scale research
2. Provide a variety of image analysis
3. Use a dedicated analysis approach to track multiple cellular processes
4. Observe the overall characteristics of the cell population
5. High throughput
6. Multiple indicators simultaneous detection
7. Saving samples
8. Detectable cells, tissue

1. Multi-technology combination, evaluation indicators increased
2. Mutual evidence to ensure accurate data

Disadvantages

1. Require professional technicians to operate
2. Low success rate
3. Long time
4. Low efficiency
5. Only detect electrical signal changes

1. Low sensitivity
2. The instrument is expensive
3. The detection object can only be a cell
4. Only detect changes in electrical signals

1. High requirements on the state and quality of the test object

1. Implementation is cumbersome
2. Long time
3. Waste of samples
4. Loss reagent